Assessment of transient expression strategies to sialylate recombinant proteins in N. benthamiana

There is a demand for increasing the current manufacturing capacities for recombinant protein-based drugs. Novel expression systems such as plants are being explored as faster, more flexible, and possibly cheaper plat-forms. Many of these therapeutic proteins are glycosylated and require terminal sialylation to attain full bio-logical activity. In planta protein sialylation has been achieved by the introduction of an entire mammalian biosynthetic pathway in Nicotiana benthamiana, comprising the coordinated expression of the genes for (i) biosynthesis, (ii) activation, (iii) transport, and (iv) transfer of Neu5Ac to terminal galactose. Here we address technical issues that can compromise the efficacy of protein sialylation and how they can be overcome. We used the same reporter protein to compared three strategies to transiently deliver the sialylation pathway-genes evaluating efficacy, heterogeneity and batch-to-batch consistency. In addition, we assess the ability of the single-step method to sialylated additional recombinant proteins with different complexity and number of glycosylation sites. Finally, we show that efficient protein sialylation can be up-scaled for large-scale production of sialylated proteins in plants.

Automated summary

There is a demand to increase manufacturing capacities for recombinant protein-based drugs. Novel expression systems such as plants are being explored for faster and more flexible production. In this study, we discuss the technical issues and solutions for achieving efficient protein sialylation in plants. We also evaluate different strategies for delivering the sialylation pathway genes and demonstrate the scalability of protein sialylation for large-scale production.